We will study the enzymatic repair of damage to the DNA of replicating and developing cells of the cellular slime mold, Dictyostelium discoideum, after treatment with certain mutagenic and carcinogenic agents, including ionizing radiation, ultraviolet light, and alkylating chemicals. Repair at various stages of development will be compared to that in the replicating vegetative cells. The genetics and biochemistry of repair in a series of repair-defective mutants will be characterized using parasexual genetic techniques, specific-enzyme monitoring of DNA damage, in vivo appearance and resealing of breaks in DNA, repair replication via density-shift techniques, BUdR photolysis of repair patches, and in vitro analysis of specific repair-enzyme activities extracted from these cells. Mutagenesis assays will quantitate mutation production in these strains with above agents administered during growth and development. Correlations will be made between mutagenesis and repair of specific lesions. Some emphasis will be placed on mutagenesis by: (1) ionizing radiation, highly mutagenic in this organism and capable of readily penetrating the developmental aggregates, and (2) nitrosoguanidine, an alkylating agent that produces relatively large amounts of 6O-methyl-guanine and shows preferential mutagenesis in replicating regions of DNA. Effects of repair modifiers such as caffeine and treatment fractionation on mutagenesis will be studied. In order to improve the introduction of isotopic labels, nutrients, inhibitors, and other molecules into these amoebae, we will investigate feeding and injection regimes utilizing loaded membrane vesicles.